The massive parallelization of biological assays and realization of single-molecule resolution have yielded profound advances in the ways that biological systems are characterized and monitored and the way in which biological disorders are treated. Assays are able to interrogate thousands of individual molecules simultaneously, often in real time. In particular, the combination of solid state electronics technologies to biological research applications has provided a number of important advances including, e.g., molecular array technology, i.e., DNA arrays (see, e.g., U.S. Pat. No. 6,261,776), microfluidic chip technologies (see e.g., U.S. Pat. No. 5,976,336), chemically sensitive field effect transistors (ChemFETs), and other valuable sensor technologies.
These biochemical and medical assays often rely on the positioning of individual assay components on a molecular scale. Thousands of nanoscale assays are often patterned on a substrate for macro-manipulation, analysis, and data recording. Accordingly, new tools are needed to arrange and construct assay components with accuracy and precision at a molecular resolution.
Zero Mode Waveguides
In some assays, molecules are confined in a series, array, or other arrangement of small holes, pores, or wells, for example, a zero mode waveguide (ZMW). ZMW arrays have been applied to a range of biochemical analyses and have found particular usefulness for genetic analysis. ZMWs typically comprise a nanoscale core, well, or opening disposed in an opaque cladding layer that is disposed upon a transparent substrate, e.g., a circular hole in an aluminum cladding film deposited on a clear silica substrate. J. Korlach et al., Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. 105 PNAS 1176-81 (2008). A typical ZMW hole is ˜70 nm in diameter and ˜100 nm in depth. ZMW technology allows the sensitive analysis of single molecules because, as light travels through a small aperture, the optical field decays exponentially inside the chamber. That is, due to the narrow dimensions of the well, electromagnetic radiation that is of a frequency above a particular cut-off frequency will be prevented from propagating all the way through the core. Notwithstanding the foregoing, the radiation will penetrate a limited distance into the core, providing a very small illuminated volume within the core. By illuminating a very small volume, one can potentially interrogate very small quantities of reagents, including, e.g., a single molecule and single molecule reactions. The observation volume within an illuminated ZMW is ˜20 zeptoliters (20×10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single nucleotide can be readily detected.
By monitoring reactions at the single molecule level, one can precisely identify and/or monitor a given reaction. The technology is not limited in the types of single molecule interactions that can be observed (e.g., a non-limiting list is protein-protein, protein-DNA, DNA-DNA, DNA-RNA, RNA-RNA, protein-RNA, lipid-lipid, protein-lipid, enzyme-substrate, enzyme-intermediate, enzyme-product, enzyme-metabolite, enzyme-cofactor, enzyme-inhibitor, etc.). In particular, the technology is the basis for a particularly promising field of single molecule DNA sequencing that monitors the molecule-by-molecule (e.g., nucleotide-by-nucleotide) synthesis of a DNA strand in a template-dependent fashion by a single polymerase enzyme (e.g., Single Molecule Real Time (SMRT) DNA Sequencing as performed, e.g., by a Pacific Biosciences RS Sequencer (Pacific Biosciences, Menlo Park, Calif.)). See, e.g., U.S. Pat. Nos. 7,476,503; 7,486,865; 7,907,800; and 7,170,050; and U.S. patent application Ser. Nos. 12/553,478, 12/767,673; 12/814,075; 12/413,258; and 12/413,466, each incorporated herein by reference in its entirety for all purposes. See also, Eid, J. et al. 2009. “Real-time DNA sequencing from single polymerase molecules”, 323 Science: 133-38 (2009); Korlach, J. et al. “Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides”, 27 Nucleosides, Nucleotides & Nucleic Acids: 1072-82 (2008); Lundquist, P. M. et al., “Parallel confocal detection of single molecules in real time”, 33 Optics Letters: 1026-28 (2008); Korlach, J. et al., “Selective aluminum passivation for targeted immobilization of single dna polymerase molecules in zero-mode waveguide nanostructures”, 105 Proc Natl Acad Sci USA: 1176-81 (2008); Foquet, M. et al., “Improved fabrication of zero-mode waveguides for single-molecule detection”, 103 Journal of Applied Physics (2008); and Levene, M. J. et al. “Zero-mode waveguides for single-molecule analysis at high concentrations”, 299 Science: 682-86 (2003), each incorporated herein by reference in its entirety for all purposes.
In conventional use, placing components in the wells of the ZMW relies on simple diffusion to deliver components (e.g., macromolecules such as DNA polymerase and/or DNA and/or DNA/DNA polymerase complexes) to the desired site (e.g., near the opening or to the bottom of the ZMW well) in the zero mode waveguides. As a result, a significant amount of the macromolecule (e.g., the DNA polymerase/DNA complex) needs to be added to the ZMWs to achieve a critical mass sufficient to promote the diffusion of the complexes to a site near the openings of the wells or into the bottoms of the wells. This process is not efficient: e.g., only a fraction of the complexes reaches the desired sites in the wells and incubation times (e.g., 4 hours) are required to position the assay components in the proper sites.